The objective of this project is to understand the structure-function relationship of the recently discovered tumor suppressor p16 and its close relative p15. They are specific inhibitors of human cyclin-dependent kinase 4 (Cdk4) and its close variant cdk6. The approach involves a combination of genetic, biochemical, and biophysical techniques. The Specific Aim 1 is to determine the tertiary structures of p16 and p15 by NMR in combination with 15N/13C isotope labeling. The Specific Aim 2 s to identify key residues in p16 for their binding to cdk4 by site-specific mutagenesis, starting with the p16 mutants existing in tumor cells. The mutants showing no global conformational change will be analyzed for activity on the basis of their inhibition of, and binding to cdk4. The results, along with the tertiary structures determined by NMR, will allow formulation of a "working model" for the possible cdk4 binding motif for each tumor suppressor. The working models will be further refined in Specific Aim 3 by rational design of mutants. The goals are: to provide information for rational design of new cdk4 inhibitors as potential anticancer drugs (which will be the goal of the second project period), and to address whether p16 and p15 have identical cdk4 binding sites. The Specific Aim 4 is to change the specificity of p16 and/or p15 from cdk4 to cdk1 by an in vitro molecular evolution technique in combination with the yeast two-hybrid system and a phage-display assay as the screening methods. The tertiary structures of the proteins with altered specificity will be determined and compared with those of the native p16 and p15; the information will be useful in understanding the nature of the cdk specificity of tumor suppressors. The Specific Aim 5 is to search for new peptide inhibitors of cdk4 from epitope libraries of p16 or p15 and a random combinatorial library, using a two-step screening process: the two- hybrid system or phage-display, and competition with p16 or p15 for inhibition of the cdk4 activity. The new inhibitors and their peptide- mimetic or non-peptide analogs (to be designed later) are potential anticancer drugs. The Specific Aim 6 is to search for a peptide mimic of cdk4 capable of binding to p16 or p15 from epitope and combinatorial libraries. In addition to the screening methods based on binding, structural analysis by NMR will be further used to determine whether the peptide binding site agrees with the cdk4 binding site deduced in specific aims 1-3. The goal is to provide a structural model for the suppressor cdk4 complex, which will be useful in deciphering the "missing functionality" in the disease-related mutants of tumor suppressors, an important information for drug design.